Characterization of two-photon point spread function in turbid medium by direct measurements, multicolor imaging, and blind deconvolution
Journal
Proceedings of SPIE - The International Society for Optical Engineering
Journal Volume
4262
Pages
73-81
Date Issued
2001
Author(s)
Abstract
Over the past decade, scanning fluorescence microscopy based on two-photon excitation has become an important branch of microscopic bio-imaging. Compared to traditional scanning techniques, two-photon microscopy offers a number of distinct advantages. First, scanning of the point-like excitation spot used for imaging results in images with excellent axial depth discrimination. In addition, the limited extent of the excitation volume also limits specimen photo-damage to the focal volume. Finally, the long, near-infixed wavelengths used for sample excitation allow in-depth, non-invasive imaging of optically turbid biological samples. For in-depth imaging, the microscopic objective and often optically heterogeneous biological specimen forms a complex system. To optimize imaging quality in two-photon microscopy, an understanding of the point-spread-function (PSF) is essential. In this work, we attempted to characterize the two-photon PSF by two methods: direct imaging of 0.1 ym fluorescent microspheres and multicolor imaging of 2 ym green fluorescent microspheres in a uniform blue fluorescent background. In both measurements, the turbidity of the surrounding medium was varied by changing the concentration of Liposyn III, a scattering component in the specimen. We found that at discrete Liposyn III concentrations between 0 and 2%, the PSF widths were not affected by the amount of scatterers present. However, the imaged contrast continued to degrade as a function of the amount of scatter. This suggests that the broadening of the tail region of the PSF can be the cause of image contrast loss. We will also discuss the possibility of using blind- deconvolution as a method to obtain PSF information in complex biological specimen.
Type
journal article